Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Subsp. and Differentiation from Subsp. on Blueberry.

Bacterial leaf scorch (BLS), caused by (), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by subsp. (). However, a recent survey revealed the presence of another subspecies, subsp. (), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor () gene sequence of to discriminate between the two subspecies infecting blueberry. With the CAPS marker, specific detection of isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the gene PCR product (amplified with primers RST31 and RST33) using the enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the gene from both pure bacterial cultures and infected plant material using the Genie III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.